ATP-dependent restriction enzyme SauUSI from Staphylococcus aureus is also a bona fide single strand DNA endonuclease

Restriction endonucleases cleave exogenous DNA thus restricting horizontal gene transfer and phage infection of host bacterium. This nucleolytic activity occurs on double-stranded DNA (dsDNA) and is target site specific. Here we report that the Type IV ATP-dependent restriction endonuclease SauUSI from Staphylococcus aureus also possesses a hitherto unknown single-stranded DNA (ssDNA) endonuclease activity. We demonstrate that, unlike the dsDNA cleavage activity, ssDNA cleavage by SauUSI does not require divalent cation or ATP hydrolysis and is target-site and DNA methylation-status independent. Furthermore, we show that SauUSI can cut ssDNA gaps, overhangs, bubbles and loops but not ssRNA. The activity is inhibited at higher concentrations of magnesium ion, ATP, and the presence of single strand DNA binding protein. The ssDNA nuclease activity is thus tightly regulated and may protect the host DNA from damage by SauUSI. ### Competing Interest Statement The authors have declared no competing interest.